Cell death is a fundamental aspect of animal development. A considerable proportion of the cells that are generated die during the normal development of both vertebrates (Glucksmann, Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1951)) and invertebrates (Truman and Schwartz, Ann. Rev. Neurosci. 7:171-188 (1984)). Cell death plays a role in morphogenesis (e.g., of the eye, secondary palate, heart, nervous system and limbs in vertebrate embryos), metamorphosis (e.g., in moths and other insects), and tissue homeostasis (e.g., of epithelial linings and the thymus), as well as in neuron selection during the establishment of synaptic connections and in sexual dimorphism (reviewed by Ellis et al., Ann. Rev. Cell Biol. 7:663-698 (1991)). Cell death which occurs as a part of normal development will be referred to herein as physiological cell death.
Besides physiological cell death, cell death may occur as a pathological manifestation of disease, in which case it will be referred to herein as pathological cell death (see review by Trump and Mergner (1974), in: The Inflammatory Process, vol. 1, 2nd ed. (eds. Zweifach et al.), Academic Press, New York, pp. 115-257). Cell death can result from a variety of injuries to the cell, including toxins, ischemia (lack of blood supply), hypoxia (lack of oxygen) and infectious agents, as well as from genetic mutations. The major clinical aspects of most degenerative diseases are a consequence of cell death. For example, Huntingtons""s disease, Parkinson""s disease, Alzheimer""s disease and amyotrophic lateral sclerosis are marked by degeneration of neurons, while Duchenne muscular dystrophy is characterized by muscle degeneration. In addition, some cancers are thought to be caused by a defect in cell death processes. Thus, understanding and preventing cell death can be viewed as one of the major goals of biomedical research.
The simple and invariant anatomy and development of the nematode Caenorhabditis elegans have made it an attractive system for the study of cell death. Because C. elegans is small, cellularly simple and transparent, Nomarski differential interference microscopy can be used to observe individual cells throughout development. As a result, the complete cell lineage of C. elegans, from zygote to adult, has been elucidated (Sulston and Horvitz, Dev. Biol. 82:110-156 (1977); Kimble and Hirsh, Dev. Biol. 70:396-417 (1979); Sulston et al., Dev. Biol. 100:64-119 (1983)).
Cell death is an important component of the development of C. elegans: during the development of the adult hermaphrodite, the generation of 816 nongonadal cells is accompanied by the generation and subsequent deaths of an additional 131 cells. Cell death appears to be an integral part of the differentiation of a variety of tissues. The pattern of cell deaths is essentially invariant among different animals, i.e, the same set of cells die at the same developmental time. In addition, a vast majority of cell deaths in C. elegans does not appear to be initiated by interaction with surrounding cells or diffusible factors.
Genetic analysis has identified many genes that affect programmed cell death in C. elegans (reviewed by Ellis et al. (1991) supra). The activities of two genes, ced-3 and ced-4, seem to be required for the onset of almost all C. elegans programmed cell deaths (Ellis and Horvitz, Cell 44:817-829 (1986)). Mutations in ced-3 and ced-4 block essentially all programmed cell deaths. In ced-3 and ced-4 mutants, cells that normally undergo programmed cell death instead survive, differentiate and even function (Ellis and Horvitz (1986) supra; Avery and Horvitz, Cell 51:1071-1078 (1987); White et al., Phil. Trans. R. Soc. Lond. B. 331:263-172 (1991)). Genetic analyses indicate that ced-3 and ced-4 genes most likely act within dying cells; this suggests that of these genes are expressed within dying cells and either encode cytotoxic molecules or control the activities of cytotoxic molecules (Yuan and Horvitz, Dev. Biol. 138:33-41 (1990)).
Relatively little is known about the mechanism of cell death. Initiation of cell death occurs in response to a variety of signals. External injuries and cytotoxic agents cause cells to die. Endocrine signals trigger cell death during insect metamorphosis, thymocyte death and regression of the prostate in the male rat after castration. Lack of neuronal growth factors is suspected to be the cause of certain neuronal cell deaths during vertebrate development and may also be the cause of cell deaths in certain neurodegenerative diseases. A specific protein, Mullerian inhibiting substance, is responsible for the regression of the Mullerian duct during the development of male mammals. In addition, genetically programmed cell deaths which occur apparently autonomously of cellxe2x80x94cell interaction or diffusible factors are observed in C. elegans and other invertebrates. (Truman and Schwartz, Neuro. Comm. 1:66-72 (1982); Cohen and Duke, J. Immunol. 132:38-42 (1984); Isaacs, Prostate 5:545-557 (1984); Martin et al., J. Cell. Biol. 106:829-844 (1988); Oppenheim and Prevette, Neurosci. Abstr. 14:368 (1988); Beal et al., Nature 321:168-171 (1986); Birkmayor and Hornykiewicz, Advances in Parkinsonism, Fifth International Symposium on Parkinson""s Disease, Vienna, Roche, Basle, 1976; Lagsto et al., Science 219:979-980 (1983); Rossor, Lancet 2:1200-1204 (1982); Biel et al., Science 229:289-291 (1985); Cosi et al., in: Advances in Experimental Medicine and Biology, vol. 209, Plenum Press, New York, 1987; Bonilla et al., Cell 54:447-452 (1988); Picard and Josso, Biomedicine 25:147-150 (1976)).
Cell deaths also vary morphologically. Two major categories of cell deaths have been established based on morphological features (Kerr et al., Br. J. Cancer 26:239-257 (1972)). The first type of cell death, called necrosis, is characterized by cellular swelling, rupture of plasma and internal membranes, and eventual leakage of cellular contents into the extracellular space. The second, called apoptosis, involves progressive condensation of cytoplasm and nuclear chromatin and eventual fragmentation of cellular membranes into xe2x80x98apoptotic bodiesxe2x80x99, which are usually digested by macrophages or adjacent epithelial cells. Necrosis is most often a manifestation of certain pathological conditions, e.g., injury by complement (Hawkins et al., Am. J. Pathol. 68:255-288 (1972)), hypoxia (Jennings et al., Am. J. Pathol. 81:179-198 (1975)), or exposure to a variety of toxins (McLean et al., Int. Rev. Exp. Pathol. 4:127-157 (1965)). In contrast, apoptosis is usually associated with physiological conditions, e.g., embryogenesis (Bellaris, J. Anat. 95:54-60 (1961); Saunders, Science 154:604-612 (1966)) and metamorphosis (Truman, Ann. Rev. Neurosci. 7:171-188 (1984). Interestingly, morphological features of physiological cell death in C. elegans resemble, in general, those of apoptosis in vertebrates (Ellis et al., Ann. Rev. Cell Biol. 7:663-698 (1991)). However, deviations from the standard descriptions of necrosis and apoptosis are often observed. It is uncertain whether this morphological classification reflects real differences in underlying mechanisms of cell death.
Although the initiation and morphology of cell death vary, there is evidence which suggests that most physiological and some pathological cell deaths may share a common feature involving the activation of cell death genes. The existence of a genetic cell death program in a variety of organisms is suggested by the observation that protein and RNA synthesis inhibitors can prevent or delay a variety of cell deaths (insect metamorphosis, prostate regression, vertebrate neuronal cell death and thymocyte cell death) (Lockshin, J. Insect Physiol. 15:1505-1516 (1969); Stanisic et al., Invest. Urol. 16:19-22 (1978); Martin et al. (1988) supra; Oppenheim and Prevette (1988) supra; Cohen and Duke (1984) supra), New RNA and protein species have been found after the initiation of cell death in the rat prostate after castration (Buttyan et al., Molecular Endocrinology 2:650-657 (1988); Lee et al., Prostate 7:171-185 (1985)). Thus, a better understanding of the mechanisms of cell death would have wide biological application and provide a basis for altering or controlling the process.
The present invention relates to genes, referred to herein as cell death-protective genes, which function to protect cells against programmed cell death by antagonizing the activity of genes which cause cell death. As described herein, Applicants have identified what appears to be a key or master regulatory gene whose activity determines whether a cell survives or undergoes cell death.
Specifically, a cell death-protective gene from the nematode Caenorhabditis elegans, called ced-9, has been identified, sequenced, and characterized. ced-9 is essential for C. elegans development and apparently functions by protecting cells which normally live during development from programmed cell death. As is also described herein, a mutation that constitutively activates ced-9 prevents cells which normally die during development from undergoing programmed cell death, and mutations that inactivate ced-9 result in the deaths of cells which normally survive during development and consequently, in embryo lethality. ced-9 has been shown to function by antagonizing the activities of the cell death genes ced-3 and ced-4. Thus, the C. elegans ced-9 gene appears to act as a binary switch to regulate programmed cell death. Results described herein indicate that many and possibly all cells that survive during C. elegans development do so because ced-9 activity prevents them from undergoing programmed cell death.
In addition, a human equivalent of the C. elegans ced-9 gene has been discovered. The deduced amino acid sequence of the ced-9 gene product was found to have about 23% identity and about 47% similarity to the product of the human oncogene bcl-2. This structural similarity, together with previous studies on bcl-2 activity in lymphocytes, strongly suggests that bcl-2 is a human equivalent of ced-9. Applicants further provide methods for identifying other cell death-protective genes from a variety of organisms, including vertebrates (e.g., mammals and particularly humans), invertebrates (e.g., insects), microbes (e.g., yeast), and possibly plants. Furthermore, comparison of ced-9, bcl-2, and other cell death-protective genes and their encoded products provides a way to define key functional features or regions of these genes and gene products. Those features or parts that are conserved between these genes or their gene products are most likely to be functionally important.
Applicants further provide methods and agents for altering the occurrence of cell death in a population of cells and hence, affecting the proliferative capacity and longevity of tissues or organisms. Methods and agents for both decreasing and increasing cell deaths are described. The agents may be all or portions of the cell death-protective genes and encoded products, or derivatives, mimetics, activators or inactivators, or agonists or antagonists of the activity of cell death-protecting genes.
As a result of this work, methods and agents for altering cell death are available for therapeutic or preventive treatment of diseases or conditions involving cell death. Methods and agents for reducing cell death are available and are potentially useful for treating disorders and conditions, including those associated with aging, stroke, traumatic brain injury, myocardial infarction, degenerative diseases (including Huntington""s disease, amyotropic lateral sclerosis, Alzheimer""s disease, Parkinson""s disease, and Duchenne""s muscular dystrophy), and viral and other types of infection (such as with the human immunodeficiency virus or HIV). Methods and agents for increasing cell deaths are also available which are potentially useful for decreasing the growth of or for killing specific cell populations, such as infected cells or autoreactive immune cells. These methods and agents may also be useful for treating diseases or conditions characterized by excessive cell growth or an abnormally low frequency of cell death (e.g., neoplasia and other cancerous growth). Methods and agents which increase cell death are also potentially useful for treating viral, parasitic, and other infections and to kill undesirable organisms, for example, in pest control or biological containment applications.
Accordingly, in one aspect, the invention features substantially pure nucleic acid which is a cell death-protective gene. Preferably, the cell death-protective gene is the C. elegans ced-9 gene. In a related aspect, the invention features substantially pure nucleic acid consisting essentially of all or a portion of the nucleotide sequence selected from the group consisting of: a) the nucleotide sequence shown in FIG. 2; b) the nucleotide sequence shown in FIG. 3; and c) substantially pure nucleic acid encoding the amino acid sequence shown in FIG. 4. In further related aspect, the invention includes isolated protein encoded by the C. elegans ced-9 gene, preferably consisting essentially of the amino acid sequence shown in FIG. 4; and an antibody specifically reactive with the protein encoded by the C. elegans ced-9 gene.
In another aspect, the invention features substantially pure nucleic acid which is a mutated cell death-protective gene, wherein the mutation constitutively activates the gene. In one embodiment, the mutated cell death-protective gene is C. elegans ced-9, more preferably, the mutation is n1950 and, most preferably, the mutation is associated with a glycine to glutamic acid change at codon 169. In another embodiment, the constituatively activating mutation in the cell death-protective gene is human bcl-2. Preferably, the mutation is associated with a glycine to glutamic acid change at codon 145.
In additional aspects, the invention features substantially pure nucleic acid which is a mutated cell death-protective gene, wherein the mutation inactivates the gene and isolated protein encoded by this nucleic acid. In one embodiment the cell death-protective gene is C. elegans ced-9, and, preferably, the mutation is selected from the group consisting of: a) n2077; b) n2161; and c) n1653ts; or, the mutation is associated with a change selected from the group consisting of: a) tyrosine to asparagine at codon 149; and b) glutamine to termination at codon 160. In another embodiment, the cell death-protective gene having a mutation which inactivates the gene is human bcl-2.
In another aspect, the invention features a method for identifying a novel cell death-protective gene by the steps of: a) obtaining a structurally similar gene by: 1) identifying a gene which is structurally similar to a cell death-protective gene; or 2) identifying a gene whose gene product is similar to the gene product of a cell death-protective gene; and b) determining that the structurally similar gene protects cells in which it functions from cell death. Identification of this similarity indicates a novel cell death-protective gene.
In one embodiment of this method for identifying genes, step a) includes the steps of: i) combining DNA with a nucleic acid probe comprising the cell death-protective gene, or a portion able to specifically hybridize to the cell death-protective gene, under conditions suitable for specific hybridization of the nucleic acid probe to complementary sequences; and ii) detecting specific hybridization of the nucleic acid probe to the DNA, wherein specific hybridization indicates that a structurally similar gene, or portion, is present in the DNA. Preferably, the DNA used in this embodiment is a gene library; or the nucleic acid probe further comprises degenerate oligonucleotides derived from the amino acid sequence of the product of the cell death-protective gene. In another embodiment, step a) includes the steps of: i) combining nucleic acid with primers comprising portions of the cell death-protective gene under conditions suitable for polymerase chain reaction; and ii) detecting specific DNA amplification, wherein specific DNA amplification produces a structurally similar gene, or portion. Preferably, the primers used in this embodiment further include degenerate oligonucleotides derived from the amino acid sequence of the product of the cell death-protective gene.
In another embodiment, step a) includes the steps of: i) combining an expression gene library with an antibody which binds specifically with the protein encoded by the cell death-protective gene under conditions suitable for specific antibody-antigen binding of the antibody to antigens expressed from the gene library; and ii) detecting specific antibody-antigen binding, wherein specific antibody-antigen binding indicates that a structurally similar gene is present in the expression gene library, thereby identifying a gene which is structurally similar to a cell death-protective gene.
In additional embodiments, step a) comprises searching a database of genes for a nucleotide sequence which is similar to the nucleotide sequence of the cell death-protective gene or searching a database of proteins for an amino acid sequence which is similar to the amino acid sequence of the protein encoded by the cell death-protective gene.
In yet another embodiment, step b) includes the steps of: i) using the structurally similar gene and a nematode which lacks the activity of the ced-9 gene to produce a transgenic nematode; and ii) determining a decrease in the cell deaths which occur during the development of the transgenic nematode relative to the nontransgenic nematode. A decrease in cell deaths indicates that the structurally similar gene protects cells in which it functions from cell death.
In another embodiment, step b) includes the steps of: i) introducing the structurally similar gene into cultured mammalian cells to produce transfected cells which express the gene; and ii) determining a decrease in cell deaths among the transfected cells under conditions which induce cell death, wherein a decrease in cell deaths indicates that the gene protects cells in which it functions from cell death.
In another aspect, the invention features a bioassay for identifying a gene which has cell death-protective activity. This bioassay includes the steps of: a) using DNA and a nematode which lacks the activity of the ced-9 gene to produce a transgenic nematode; and b) determining a decrease in the cell deaths which occur during the development of the transgenic nematode relative to the nontransgenic nematode. A decrease in cell deaths indicates the activity of a cell death-protective gene in the DNA, and thereby allows identification of a gene which has cell death-protective activity. Preferably, the nematode used in the bioassay underexpresses ced-9 or expresses inactivated Ced-9 protein; and/or the DNA is from an organism other than a nematode; and/or the DNA is an expression gene library. In a related aspect, the invention features substantially pure nucleic acid which is a cell death-protective gene identified by the foregoing bioassay and mutated derivatives thereof.
In another aspect, the invention features a bioassay to identify a mutation in a cell death-protective gene which alters the activity of the gene, comprising the steps of: a) using a cell death-protective gene having a mutation and a nematode which lacks ced-9 activity to produce a transgenic nematode; and b) comparing cell deaths which occur during the development of the transgenic nematode having the mutated gene with those which occur in a transgenic nematode having a non-mutated gene, wherein a difference in cell deaths indicates that the mutation alters the activity of the cell death-protective gene. This alteration in activity indicates a mutation in a cell death-protective gene which alters the activity of the gene. In a related aspect, the invention features substantially pure nucleic acid which is a cell death-protective gene having a mutation identified by this bioassay.
In yet another aspect, the invention features a bioassay for identifying an agent which mimics the activity of a cell death-protective gene, comprising the steps of: a) introducing an agent into a nematode which lacks the activity of ced-9; and b) detecting a decrease in cell deaths which occur in the nematode, wherein a decrease indicates that the agent mimics the activity of a cell death-protective gene. Preferably, the nematode used in the bioassay underexpresses ced-9 or expresses inactivated Ced-9 protein; and/or the agent is introduced into the nematode by a method selected from: microinjection, diffusion, or ingestion. In a related aspect, the invention features an agent identified by this bioassay.
In another aspect, the invention features a bioassay for identifying an agent which affects the activity of a cell death-protective gene, and includes the steps of: a) introducing an agent into a nematode which expresses a cell death-protective gene; and b) detecting a change in the pattern of cell deaths which occur in the development of the nematode, wherein a change indicates that the agent affects the activity of the cell death-protective gene. Preferably, the cell death-protective gene used in the assay is ced-9; or the nematode is a transgenic nematode produced from the integration of cell death-protective gene (e.g., bcl-2) into a nematode which lacks ced-9 activity. The nematode used in this may overexpresses or underexpresses the cell death-protective gene, or expresses an inactivated or constitutively activated form of the cell death-protective gene.
In a related aspect, the invention features an agent which affects the activity of a cell death gene identified by this bioassay. Preferably, the agent is selected from the group consisting of: a) single stranded nucleic acid comprising all or a portion of the antisense sequence of a cell death-protective gene which is complementary to the mRNA encoded by the gene; and b) DNA encoding a). The agent may also be a gene which is not a cell death-protective gene, or may be a mutated cell death-protective gene which does not protect cells from cell death and which antagonizes the activity of cell death-protective genes.
In another aspect, the invention features a bioassay for identifying a cell death-protective gene, which includes the steps of: a) introducing DNA into cultured mammalian cells to produce transfected cells which express gene(s) in the DNA; and b) detecting cell deaths among the transfected cells under conditions which induce cell death, wherein a decrease in cell deaths indicates the activity of a cell death-protective gene in the DNA. Preferably, the DNA used in the bioassay is an expression gene library. In a related aspect, the invention features substantially pure nucleic acid identified in the bioassay.
In another aspect, the invention features a bioassay for identifying a mutation in a cell death-protective gene which alters the activity of the gene and includes the steps of: a) introducing a cell death-protective gene having a mutation into cultured mammalian cells to produce transfected cells which express the mutated gene; and b) comparing cell deaths which occur among the transfected cells under conditions which induce cell death to cell deaths which occur among cells transfected with the non-mutated gene. A difference in cell deaths indicates that the mutation alters the activity of the gene. In a related aspect, the invention features substantially pure nucleic acid which is a cell death-protective gene having a mutation identified by the bioassay.
In a further aspect, the invention features a bioassay for identifying an agent which mimics the activity of a cell death-protective gene and includes the steps of: a) exposing cultured mammalian cells to an agent under conditions which induce cell death and which are appropriate for activity of the agent; and b) detecting cell deaths in said exposed cells, wherein a decrease in cell deaths indicates that the agent protects cells from cell death. In a related aspect, the invention features an agent identified by this bioassay.
In yet another aspect, the invention features a bioassay for identifying an agent which affects the activity of a cell death-protective gene and includes the steps of: a) exposing cultured mammalian cells which are protected from conditions which induce cell death by the activity of a cell death-protective gene to an agent under conditions which are appropriate for activity of the agent; and b) detecting cell deaths in the exposed cells. A change in cell deaths by this method indicates that the agent affects the activity of the gene. In a related aspect, an invention features an agent identified by this bioassay.
In another aspect, the invention features a method for altering cell death in a cell by altering in the cell the activity of a cell death-protective gene. Preferably, the cell death-protective gene is ced-9 or bcl-2 and/or the cell is a nematode cell or a human cell. In certain embodiments, the cell death is prevented by activation of the cell death-protective gene expression of the cell death-protective gene by virtue of a mutation which activates the protective gene product. The mutation may be the n1950 mutation in ced-9, or another mutation which is associated with a change from glycine to glutamic acid at codon 169, or the mutation may be a mutation in bcl-2, e.g., a change from glycine to glutamic acid at codon 145. In another embodiment, cell death is caused. This method involves inactivation of the cell death-protective gene so that it does not provide protection against programmed cell death. Preferably, cell death is caused by inactivating a cell death gene by mutation. For example, a mutation in ced-9 or bcl-2 may be employed. Where the cell death-protective gene is C. elegans ced-9, the mutation may be selected from the group consisting of: a) n2077; b) n2161; and c) n1653ts. The method of altering cell death may further include exposing the cell to an agent which alters the activity of a cell death-protective gene in the cell under conditions appropriate for activity of the agent.
Where the method of altering cell death involves increasing the activity of the cell death-protective gene, the method may include exposing the cell to an agent selected from the group consisting of: a) DNA which includes the cell death-protective gene; b) RNA encoded by the cell death-protective gene; c) protein encoded by the cell death-protective gene, or active portion thereof; d) an agent which mimics the activity of the cell death-protective gene; e) an activator of the cell death protective gene; and f) an agonist of the cell death-protective gene. Where the method involves decreasing the activity of the cell death-protective gene, the method may include exposing the cell to an agent selected from the group consisting of: a) single stranded nucleic acid which is complementary to the mRNA of the cell death-protective gene; b) DNA which directs the expression of a); c) a mutated cell death-protective gene which does not protect from cell death and which antagonizes the activity of the cell death-protective gene; d) an inactivator of the cell death-protective gene; and e) an antagonist of the cell death-protective gene.
In another aspect, the invention features a method for treating a condition characterized by increased cell deaths in a tissue. The method includes increasing activity of a cell death-protective gene which functions in the cells which are affected by the condition. Preferably, the affected tissue is exposed to an agent selected from the group consisting of: a) DNA comprising the cell death-protective gene; b) RNA encoded by the cell death-protective gene; c) protein encoded by the cell death-protective gene, or active portion thereof; d) an agent which mimics the activity of the cell death-protective gene; e) an activator of the cell death protective gene; and f) an agonist of the cell death-protective gene, under conditions appropriate for activity of the agent. The cell death-protective gene is selected from the group consisting of: a) bcl-2; b) a novel human cell death-protective gene which is structurally similar to bcl-2; and c) a novel human cell death-protective gene which is structurally similar to ced-9. The condition being treated is selected from the group consisting of: a) myocardial infarction; b) stroke; c) traumatic brain injury; d) neurodegenerative disease; e) muscular degenerative disease; f) aging; g) hypoxia; h) ischemia; i) toxemia; and j) infection; or the condition is a viral infection such as infection by the human immunodeficiency virus.
In another aspect, the invention provides a method for reducing a population of cells by decreasing the activity of a cell death-protective gene which functions in the cells. Preferably, the cells of the method are exposed to an agent selected from the group consisting of: a) single stranded nucleic acid which is complementary to the mRNA of the cell death-protective gene; b) DNA which directs the expression of a); c) a mutated cell death-protective gene which does not protect from cell death and which antagonizes the activity of the cell death-protective gene; d) an inactivator of the cell death-protective gene; and e) an antagonist of the cell death-protective gene, under conditions appropriate for the activity of the agent. Also preferably, the population of cells is selected from the group consisting of: a) neoplastic growth; b) cancerous tissue; c) infected cells; and d) autoreactive immune cells; and, preferably, the cell death-protective gene is selected from the group consisting of: a) bcl-2; b) a novel human cell death-protective gene which is structurally similar to bcl-2; and c) a novel human cell death-protective gene which is structurally similar to ced-9.
In another aspect, the invention features a method for treating a parasitic infection of a host animal, which includes the steps of administering an agent which decreases the activity of a cell death-protective gene which is specific to the parasite and does not harm the host animal. Preferably, the anti-parasitic agent is selected from: a) single stranded nucleic acid having all or a portion of the antisense sequence of the cell death-protective gene which is complementary to the mRNA of the gene; and b) DNA which encodes a); and, preferably, the parasite is a nematode.
In a further aspect, the invention provides a method of pest control, comprising decreasing the activity of a cell death-protective gene in the pest. Preferably, the method includes exposing the pest to an agent selected from the group consisting of: a) single stranded nucleic acid having all or a portion of the antisense sequence of the cell death-protective gene which is complementary to the mRNA of the gene; b) DNA which directs the expression of a); c) a mutated cell death-protective gene which does not protect from cell death and which antagonizes the activity of the cell death-protective gene; d) an inactivator of the cell death-protective gene; and e) an antagonist of the cell death-protective gene, under conditions appropriate for the activity of the agent.
In yet another aspect, the invention provides a method of biological containment of a recombinant organism by introducing nucleic acid into the organism which is able to direct the expression of an agent which inactivates a cell death-protective gene in the organism under predetermined conditions. Preferably, the agent used for containment is selected from the group consisting of: a) single stranded nucleic acid which has all or a portion of the antisense sequence of the cell death-protective gene which is complementary to the mRNA of the gene; b) DNA which directs the expression of a); c) a mutated cell death-protective gene which does not protect from cell death and which antagonizes the activity of the cell death-protective gene; and d) an inactivator of the cell death-protective gene; and more preferably, the agent kills the recombinant organism upon completion of a desired task by the organism.